DNA sequences in the chromosome are transcribed into pre-mRNAs which contain coding regions (exons) and generally also contain intervening non-coding regions (introns). Introns are removed from pre-mRNAs in a precise process referred to as splicing. In most cases, the splicing reaction occurs within the same pre-mRNA molecule, which is termed cis-splicing. Splicing between two independently transcribed pre-mRNAs is termed trans-splicing. Trans-splicing was first discovered in trypanosomes (Sutton & Boothroyd, 1986, Cell 47:527; Murphy et al., 1986, Cell 47:517) and subsequently in nematodes (Krause & Hirsh, 1987, Cell 49:753); flatworms (Rajkovic et al., 1990, Proc. Nat'l. Acad. Sci. USA, 87:8879; Davis et al., 1995, J. Biol. Chem. 270:21813) and in plant mitochondria (Malek et al., 1997, Proc. Nat'l. Acad. Sci. USA 94:553). In the parasite Trypanosoma brucei, all mRNAs acquire a splice leader (SL) RNA at their 5′ termini by trans-splicing. A 5′ leader sequence is also trans-spliced onto some genes in Caenorhabditis elegans. This mechanism is appropriate for adding a single common sequence to many different transcripts.
The mechanism of spliced leader trans-splicing, which is nearly identical to that of conventional cis-splicing, proceeds via two phosphoryl transfer reactions. The first causes the formation of a 2′-5′ phosphodiester bond producing a ‘Y’ shaped branched intermediate, equivalent to the lariat intermediate in cis-splicing. The second reaction, exon ligation, proceeds as in conventional cis-splicing. In addition, sequences at the 3′ splice site and some of the snRNPs which catalyze the trans-splicing reaction, closely resemble their counterparts involved in cis-splicing.
Trans-splicing may also refer to a different process, where an intron of one pre-mRNA interacts with an intron of a second pre-mRNA, enhancing the recombination of splice sites between two conventional pre-mRNAs. This type of trans-splicing was postulated to account for transcripts encoding a human immunoglobulin variable region sequence linked to the endogenous constant region in a transgenic mouse (Shimizu et al., 1989, Proc. Nat'l. Acad. Sci. USA 86:8020). In addition, trans-splicing of c-myb pre-RNA has been demonstrated (Vellard, M. et al. Proc. Nat'l. Acad. Sci., 1992 89:2511-2515), trans-spliced RNA transcripts from SV40 have been detected in cultured cells and nuclear extracts (Eul et al., 1995, EMBO. J. 14:3226) and more recently, the transcript from the p450 gene in human liver has been shown to be trans-spliced (Finta et al., 2002, J. Biol Chem 22:5882-5890). However, in general, naturally occurring trans-splicing of mammalian pre-mRNAs is thought to be an exceedingly rare event.
In vitro trans-splicing has been used as a model system to examine the mechanism of splicing by several groups (Konarska & Sharp, 1985, Cell 46:165-171 Solnick, 1985, Cell 42:157; Chiara & Reed, 1995, Nature 375:510). Reasonably efficient trans-splicing (30% of cis-spliced analog) was achieved between RNAs capable of base pairing to each other, splicing of RNAs not tethered by base pairing was further diminished by a factor of 10. Other in vitro trans-splicing reactions not requiring obvious RNA-RNA interactions among the substrates were observed by Chiara & Reed (1995, Nature 375:510), Bruzik J. P. & Maniatis, T. (1992, Nature 360:692) and Bruzik J. P. and Maniatis, T., (1995, Proc. Nat'l. Acad. Sci. USA 92:7056-7059). These reactions occur at relatively low frequencies and require specialized elements, such as a downstream 5′ splice site or exonic splicing enhancers.
U.S. Pat. Nos. 6,083,702, 6,013,487 and 6,280,978 describe the use of PTMs to mediate a trans-splicing reaction by contacting a target precursor mRNA to generate novel chimeric mRNAs. The resulting RNA can encode any gene product including a protein of therapeutic value to the cell or host organism, a toxin, such as Diptheria toxin subunit A, which causes killing of the specific cells or a novel protein not normally present in cells. The PTMs can also be engineered for the production of chimeric proteins including those encoding reporter molecules useful to image gene expression in vivo in real time or to add peptide affinity purification tags which can be used to purify and identify proteins expressed in a specific cell type.
Photodynamic therapy (PDT) of cancer uses light excitation of a photosensitive substance to produce oxygen-related cytotoxic intermediates, such as singlet oxygen or free radicals (Dougherty et al., 1993, Photochem. Photobiol. 58:895-900; Hopper et al., 2000, Lancet Oncol. 1:212-219; Ochsner et al., 1997, J. Photochem. Photobiol. B. Biol 39:1-18, Fuchs et al., 1998, Biol. Med. 24:835-847). For example, the use of CL4 for the excitation of the photosensitizer hypercin has been used for the in vitro inactivation of the equine infectious anemia virus (Carpenter, S. et al. 1994, Proc. Natl. Acad. Sci. USA 91:12273-12277). Additionally, Theodossis et al., described the in vitro photodynamic effect of rose bengal activated by intracellular generation of light generated by the oxidation of the chemiluminescent substrate luciferin, in luciferase-transfected NIH 3T3 murine fibroblasts (Theodossis et al., 2003, Cancer Research 63:1818-1821).
PDT involves the use of two individual components that combine to induce cytotoxicity in an oxygen dependent manner. The first component of PDT is a photosensitizer molecule that usually enters cells and/or tissues non-specifically. The second component involves the localized administration of light of a specific wavelength that is capable of activating the photosensitizer. Once activated the photosensitizer transfers energy from the light to molecular oxygen, thereby generating reactive oxygen species (ROS), such as singlet oxygen and free radicals. Such ROS mediate cellular toxicity. Photosensitizers may also undergo photochemical reactions that do not use oxygen as an intermediate, such as compounds that result in photoaddition to DNA. As used herein, the term photosensitizer includes, but is not limited to, other chemicals that are activated upon exposure to light. Such photosensitizers are known to those skilled in the art and the examples set forth herein are non-limiting.
Although photodynamic therapy use is desirable because of its limited side effects, its main disadvantages are the poor accessibility of light to certain tissues and the problem of restricting the delivery of light primarily to the target cells. The present invention provides methods and compositions for targeted expression of light producing enzymes in the desired cell types and in cells that otherwise are inaccessible to light, thereby providing a method for use of photodynamic therapy for the specific destruction of targeted cells. Specifically, the invention provides PTM molecules that are designed to interact with one or more cell selective target pre-mRNA species and mediate trans-splicing reactions resulting in the generation of chimeric mRNA molecules capable of encoding light producing enzyme or protein. The expression of the light producing enzyme or protein permits activation of a co-localized photosensitizer leading to death of the selected cell. The present invention provides a system for targeting cancer cell destruction. In addition, the invention provides a system for targeting selective cell death to cells infected with pathogenic microorganisms, or, cell death in instances where the activity of a particular cell type leads to disease.